Protein kinase C inhibitors

ABSTRACT

The present invention discloses compounds that are highly isozyme selective protein kinase C beta-1 and beta-2 isozyme inhibitors. The present invention also provides a method of selectively inhibiting protein kinase C isozymes beta-1, and beta-2. As isozyme selective inhibitors of beta-1 and beta-2, the compounds are therapeutically useful in treating conditions associated with diabetes mellitus and its complications, as well as other disease states associated with an elevation of the beta-1 and beta-2 isozyme.

BACKGROUND OF THE INVENTION

Protein kinase C (PKC) consists of a family of closely related enzymesthat function as serine/threonine kinases. Protein kinase C plays animportant role in cell-cell signaling, gene expression, and in thecontrol of cell differentiation and growth. At present, there arecurrently at least ten known isozymes of PKC that differ in their tissuedistribution, enzymatic specificity, and regulation. Nishizuka Y. Annu.Rev. Biochem. 58:31-44 (1989); Nishizuka Y. Science 258: 607-614 (1992).

Protein kinase C isozymes are single polypeptide chains ranging from 592to 737 amino acids in length. The isozymes contain a regulatory domainand a catalytic domain connected by a linker peptide. The regulatory andcatalytic domains can be further subdivided into constant and variableregions. The catalytic domain of protein kinase C is very similar tothat seen in other protein kinases while the regulatory domain is uniqueto the PKC isozymes. The PKC isozymes demonstrate between 40-80%homology at the amino acid level among the group, however, the homologyof a single isozyme between different species is generally greater than97%.

Protein kinase C is a membrane-associated enzyme that is allostericallyregulated by a number of factors, including membrane phospholipids,calcium, and certain membrane lipids such as diacylglycerols that areliberated in response to the activities of phospholipases. Bell, R. M.and Burns, D. J., J. Biol. Chem., 266:4661-4664 (1991); Nishizuka, Y.Science 258: 607-614 ,(1992). The protein kinase C isozymes, alpha,beta-1, beta-2 and gamma, require membrane phospholipid, calcium anddiacylglycerol/phorbol esters for full activation. The delta, epsilon,eta, and theta forms of PKC are calcium-independent in their mode ofactivation. The zeta and lambda forms of PKC are independent of bothcalcium and diacylglycerol and are believed to require only membranephospholipid for their activation.

Only one or two of the protein kinase C isozymes may be involved in agiven disease state. For example, the elevated blood glucose levelsfound in diabetes lead to an isozyme-specific elevation of the beta-2isozyme in vascular tissues. Inoguchi et al., Proc. Natl. Acad. Sci. USA89:11059-11065 L5 (1992). A diabetes-linked elevation of the betaisozyme in human platelets has been correlated with their alteredresponse to agonists. Bastyr III, E. J. and Lu, J. Diabetes 42:(Suppl 1) 97A (1993). The human vitamin D receptor has been shown to beselectively phosphorylated by protein kinase C beta. Thisphosphorylation has been linked to alterations in the functioning of thereceptor. Hsieh et al., Proc. Natl. Acad. Sci. USA 88:9315-9319 (1991);Hsieh et al., J. Biol. Chem. 268: 15118-15126 (1993). In addition,recent work has shown that the beta-2 isozyme is responsible forerythroleukemia cell proliferation while the alpha isozyme is involvedin megakaryocyte differentiation in these same cells. Murray et al., J.Biol. Chem. 268:15847-15853 (1993).

The ubiquitous nature of the protein kinase C isozymes and theirimportant roles in physiology provide incentives to produce highlyisozyme selective PKC inhibitors. Given the evidence demonstratinglinkage of certain isozymes to disease states, it is reasonable toassume that inhibitory compounds that are selective to one or twoprotein kinase C isozymes relative to the other PKC isozymes aresuperior therapeutic agents. Such compounds should demonstrate greaterefficacy and lower toxicity by virtue of their specificity.

The art recognizes various classes of compounds as protein kinase Cinhibitors. Some of these compounds are also known to demonstratespecificity to protein kinase C. However, very little is known regardingisozyme selectivity. Studies of the PKC-selective compound,3-[1-(3-dimethylaminopropyl)-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione,suggest a slight selectivity for the calcium dependent isozymes, butfind no isozyme selectivity between alpha, beta-1, beta-2, and gamma.Toullec et al., J. Biol. Chem. 266:15771-15781 (1991). Martiny-Baron, etal., J. Biol. Chem. 268:9194-9197 (1993), tested the same compound andfound slight selectivity for isozymes, alpha and beta versus delta,epsilon, and zeta. Martiny-Baron observed no differences in theselectivity between alpha and beta-1 isozymes. Wilkinson, et al.,Biochem. J. 294: 335-337 (1993), failed to observe any high degree ofisozyme selectivity and suggest only slight selectivity for the alphaisozyme and equal inhibition of beta, gamma, and epsilon for severalspecies of bis-indolemaleimides. Therefore, despite years of research,there remains a need for therapeutically effective isozyme-selectiveinhibitors.

This invention provides compounds that are highly isozyme selective. Thecompounds selectively inhibit protein kinase C beta-1 and beta-2isozymes. Accordingly, the present invention also provides a method ofselectively inhibiting protein kinase C isozymes beta-1 and beta-2. Asisozyme selective inhibitors of beta-1 and beta-2, the compounds aretherapeutically useful in treating conditions associated with diabetesmeltitus and its complications, as well as other disease statesassociated with an elevation of the beta-1 and beta-2 isozymes.

SUMMARY OF THE INVENTION

This invention provides compounds, which selectively inhibit proteinkinase C beta-1 and beta-2 isozyme, of the Formula I: ##STR1## wherein:R₁ is of the Formula II: ##STR2## R₂ is hydrogen, alkyl, acyl,alkoxyalkyl, hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl,dialkylaminoalkyl, acylaminoalkyl, N₃ -alkyl, or an amino acid of theFormula (III): ##STR3## R₃ is H or CH₃ ; R₄ is an amino acid side chain;

X is --(CH₂)_(n) --NH--, --(CH₂)_(n) --O--, phenylene--NH--,phenylene--O--, or a bond;

P₁ is H, alkyl, or an amino protecting group; and

n is 1, 2 or 3.

The invention further provides a method of selectively inhibitingprotein kinase C beta-1 and beta-2 isozyme, which comprisesadministering to a mammal in need of such treatment a pharmaceuticallyeffective amount of a compound of the Formula I. As selectiveinhibitors, the invention further provides a method for treatingdiabetes mellitus, which comprises administering to a mammal in need ofsuch treatment a pharmaceutically effective amount of a compound of theFormula I. The invention also provides pharmaceutical formulationscomprising a compound off the Formula I associated with one or morepharmaceutically acceptable excipients, carriers, or diluents.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

As noted above, tile invention provides compounds of the Formula I whichselectively inhibit isozymes of protein kinase C.

The preferred compounds are compounds of the Formula Ia: ##STR4##wherein: R₁ is ##STR5## R₂ is amino alkyl, monoalkylamino alkyl, ordialkylamino alkyl.

Particularly preferred compounds are those wherein R₄ is H, CH₃ or##STR6## and P₁ is t-butoxycarbonyl (BOC) or benzyloxycarbonyl (CBZ).

As used herein, the term "alkyl", alone or in combinations, means astraight or branched-chain alkyl group containing from one to seven,preferably one to four, carbon atoms such as methyl, ethyl, propyl,isopropyl, butyl, secbutyl, t-butyl and pentyl. The term "C₁₋₄ alkyl" isan alkyl limited to one to four carbon atoms.

The term "alkoxy", alone or in combinations, is an alkyl covalentlybonded to the parent moiety by a --O-- linkage. Examples of alkoxygroups are methoxy, ethoxy, propoxy, isopropoxy, butoxy and t-butoxy. Analkoxyalkyl is, for example, CH₃ (CH₂)_(z) --O--(CH₂)_(z) -- wherein zis from one to seven or preferably one to four.

The acyl moiety, alone or in combination, is derived from an alkanoicacid containing a maximum of 7, preferably a maximum of 4, carbon atoms(e.g. acetyl, propionyl or butyryl) or from an aromatic carboxylic acid(e.g. benzoyl). An acylamino is, for example, CH₃ (C═O)NH--(acetylamino). Likewise, an acylaminoalkyl is CH₃ (C═O)NH(CH₂)_(z) --wherein z is from one to seven or preferably one to four.

The term "amino acid side chain" represents the variable region of thenaturally occurring amino acids, which are of the formulas: ##STR7##

The term "amino protecting group" as used herein refers to substituentscommonly employed to block or protect the amino functionality. Preferredamino-protecting groups are t-butoxycarbonyl and benzyloxycarbonyl.Other amino protecting groups are found in J. W. Barton, ProtectiveGroups in Organic Chemistry, J. G. W. McOmie, Ed., Plenum Press, NewYork, N.Y., 1973, Chapter 2, and T. W. Greene, Protective Groups inOrganic Synthesis, John Wiley and Sons, New York, N.Y., 1981, Chapter 7,herein incorporated by reference. The related term "protected amino"defines an amino group substituted with an amino protecting group aspreviously discussed.

The term "pharmaceutically effective amount", as used herein, representsan amount of a compound of the invention that is capable of selectivelyinhibiting PKC isozyme activity in mammals. The particular dose of thecompound administered according to this invention will, of course, bedetermined by a physician under the particular circumstances surroundingthe case, including the compound administered, the route ofadministration, the particular condition being treated, and similarconsiderations. The compounds can be administered by a variety of routesincluding the oral, rectal, transdermal, subcutaneous, topical,intravenous, intramuscular or intranasal routes.

The term "treating," as used herein, describes the management and careof a patient for the purpose of combating the disease, condition, ordisorder and includes the administration of a compound of presentinvention to prevent the onset of the symptoms or complications,alleviating the symptoms or complications, or eliminating the disease,condition, or disorder.

The term "isozyme selective" means the preferential inhibition ofprotein kinase C beta-1 or beta-2 isozymes over protein kinase Cisozymes, alpha, gamma, delta, epsilon, zeta, and eta. In general, thecompounds demonstrate a minimum of a eight fold differential, preferablyten fold differential, in the dosage required to inhibit PKC beta-1 orbeta-2 isozymes and the dosage required for equal inhibition of thealpha protein kinase C isozyme as measured in the PKC assay. Thecompounds demonstrate this differential across the range of inhibitionand are exemplified at tile IC₅₀, i.e., a 50% inhibition. Accordingly,the invention provides a method for selectively inhibiting the beta-1 orbeta-2 protein kinase C isozyme. A related phrase is "selectivelyinhibiting protein kinase C beta-1 and beta-2 isozymes," which refers toisozyme selective inhibition. Thus, because one needs a substantiallyhigher concentration on compound to inhibit the other protein kinase Cisozymes (e.g., Example 1 discloses 50% inhibition at a concentration of0.031 μmol/L for the beta-2 isozyme while the IC₅₀ with respect to thealpha protein kinase C isozyme is 1.4 μmol/L) a pharmaceuticallyeffective dosage of the compound inhibits beta-1 and beta-2 proteinkinase C isozymes with lower toxicity by virtue of their minimalinhibition of the other isozymes. Surprisingly, the bis-indolylmaleimide containing two amino acids (i.e., R₁ is of the Formula II andR₂ is of the Formula III) are yellow in appearance. This is a markedadvantage because a yellow compound does not interfere with testing ormonitoring of the urine of patients taking the drug.

The preparation of bis-indolemalimides of the Formula: ##STR8## whereinR₁ ' is hydrogen, and R₂ ' is hydrogen, alkyl, alkoxyalkyl,hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, oracylaminoalkyl, is disclosed in U.S. Pat. No. 5,057,614 and known in theart as evidenced by EPO 397 060 (1990) and Bit et al., J. Med. Chem.36:21-29 (1993). U.S. Pat. No. 5,057,614 is herein incorporated byreference.

The compounds of Formula I, are prepared by reacting a compound ofFormula IV with an activated amino acid of the Formula V: ##STR9##wherein P₂ is an amino protecting group; and R₄ is an amino acid sidechain.

The acylation of Compound IV with the activated amino acid (Compound V)is carried out in acetonitrile in the presence of 18-crown-6 andflourine anion under basic conditions. The compounds containing an amine(e.g. Example 2) are prepared preferably with pyridine as the base forsolubility purposes. The preferred base when the solubility of thesubstrate is not important is diisopropylethylamine. The reactionconditions and other acceptable solvents are known in the art anddescribed in Klausner, et al., J. Chem. Soc. Perkin I: 607-631 (1977);and Nakagawa, et al., J. Am. Chem. Soc., 105: 3709-3710 (1983).

The compounds on Formula V are commercially available or can be preparedby known methodology, e.g., Wolman Y et al, J Chem. Soc. C: 689 (1967);Bodanszky M et al, J. Amer. Chem. Soc., 81:2504 (1959); Sakakibara Setal, Bull. Chem. Soc. Jpn., 37: 1231 (1964)

When preparing the compounds of Formula I, wherein X is --(CH₂)_(n)--NH--, --(CH₂)_(n) --O--, phenylene--NH--, or phenylene--O--, thelinking moiety, X, is appendaged to the bis-indolylmaleimide prior tothe coupling. Thus, for example, when R₁ is ##STR10## R₁ ' isaminoalkyl. Likewise, when R₁ is ##STR11## R₁ ' is hydroxyalkyl. Thecoupling reaction when X is --(CH₂)_(n) --O-- or phenylene--O-- may bealternatively carried out with dicyclohexylcarbodiimide and4-dimethylaminopyridine under standard coupling conditions known in theart.

By virtue of their acidic moieties, the compounds of Formula I includethe pharmaceutically acceptable base addition salts thereof. Such saltsinclude those derived from inorganic bases such as ammonium and alkaliand alkaline earth metal hydroxides, carbonates, bicarbonates, and thelike, as well as salts derived from basic organic amines such asaliphatic and aromatic amines, aliphatic diamines, hydroxy alkamines,and the like. Such bases useful in preparing the salts of this inventionthus include ammonium hydroxide, potassium carbonate, sodiumbicarbonate, calcium hydroxide, methylamine, diethylamine,ethylenediamine, cyclohexylamine, ethanolamine and the like.

Because of the basic moiety, the compounds of Formula I can also existas pharmaceutically acceptable acid addition salts. Acids commonlyemployed to form such salts include inorganic acids such ashydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acid, aswell as organic acids such as para-toluenesulfonic, methanesulfonic,oxalic, parabromophenylsulfonic, carbonic, succinic, citric, benzoic,acetic acid, and related inorganic and organic acids. Suchpharmaceutically acceptable salts thus include sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caproate, hepnanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, 2-butyn-1,4-dioate,3-hexyn-2, 5-dioate, benzoate, chlorobenzoate, hydroxybenzoate,methoxybenzoate, phthalate, xylenesulfonate, phenylacetate,phenylpropionate, phenylbutyrate, citrate, lactate, hippurate,β-hydroxybutyrate, glycollate, maleate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate and the like salts.

In addition to pharmaceutically-acceptable salts, other salts areincluded in the invention. They may serve as intermediates in thepurification of compounds or in the preparation of other salts, or areuseful for the identification, characterization or purification.

The pharmaceutically acceptable salts of compounds of Formula I can alsoexist as various solyates, such as with water, methanol, ethanol,dimethylformamide, ethyl acetate and the like. Mixtures of such solyatescan also be prepared. The source of such solvate can be from the solventof crystallization, inherent in the solvent of preparation orcrystallization, or adventitious to such solvent. Such solvates arewithin the scope of the present invention.

It is recognized that various stereoisomeric forms of the compounds ofFormula I may exist; for example, R₁ introduces a chiral carbon atom.The compounds are normally prepared as racemates and can conveniently beused as such, but individual enantiomers can be isolated or synthesizedby conventional techniques if so desired. Such racemates and individualenantiomers and mixture thereof form part of the present invention.

As previously noted, the compounds of the present invention are potent,beta-1 and beta-2 isozyme selective PKC inhibitors. As such, they areuseful in the treatment of conditions associated with diabetes mellitusand its complications, as well as other disease states associated withan elevation of the beta-1 and beta-2 isozymes.

Protein kinase C beta-1 and beta-2 has been linked to diabetes. Inoguchiet al., Proc. Natl. Acad. Sci. USA 89: 11059-11065 (1992). Excessiveactivity of protein kinase C has been linked to insulin signalingdefects and therefore to the insulin resistance seen in Type IIdiabetes. Karasik, A. et al., J. Biol. Chem. 265:10226-10231 (1990);Chen, K. S. et al., Trans. Assoc. Am. Physicians 104:206-212 (1991);Chin, J. E. et al., J. Biol. Chem. 268:6338-6347 (1993). Further,studies have demonstrated a marked increase in protein kinase C activityin tissues known to be susceptible to diabetic complications whenexposed to hyperglycemic conditions. Lee, T.-S. et al., J. Clin. Invest.83:90-94 (1989); Lee, T.-S. et al., Proc. Natl. Acad. Sci. USA86:5141-5145 (1989); Craven, P. A. and DeRubertis, F. R. J. Clin.Invest. 83: 1667-1675 (1989); Wolf, B. A. et al., J. Clin. Invest.87:31-38 (1991); Tesfamariam, B. et al., J. Clin. Invest. 87:1643-1648(1991); Bastyr III, E. J. and Lu, J., Diabetes 42: (Supp 11) 97A (1993).

The ability of the compounds of the present invention to selectivelyinhibit protein kinase C beta-1 and beta-2 isozyme was determined in thePKC Enzyme assay.

PKC Enzyme Assay

PKC enzymes=alpha, beta 1, beta II, gamma, delta, epsilon, eta and zeta.

Assay components in a total volume of 250 μL are the following:

Vesicles consisting of 120 μg/mL phosphatidylserine (Avanti PolarLipids)and sufficient diacylglycerol (Avanti Polar Lipids) to activatethe enzyme to maximum activity in 20 mM HEPES buffer (Sigma, St. Louis,Mo.), pH 7.5, 940 μM calcium chloride (Sigma, St. Louis, Mo.) forassaying the alpha, beta I, beta II and gamma enzyme only, 1 mM EGTA forall the enzymes, 10 mM magnesium chloride (Sigma, St. Louis, Mo.) and 30μM (gamma-32P) ATP (DuPont). For all the enzymes either histone type HL(Worthington) or myelin basic protein is used as substrate. The assay isstarted by addition of protein kinase C enzyme incubated at 30° C. for10 minutes and stopped by adding 0.5 mL of cold trichloroacetic acid(Amresco) followed by 100 μL of 1 mg/mL bovine serum albumin (Sigma, St.Louis, Mo.). The precipitate is collected by vacuum filtration on glassfiber filters employing a TOMTEC™ filtration system and quantified bycounting in a beta scintillation counter.

Using the methodoloy described, representative compounds were evaluatedand were found to have an IC₅₀ value with respect to the beta-1 andbeta-2 isozyme of below 10 μm. The compounds are isozyme selective,i.e., the compounds preferentially inhibit protein kinase C beta-1 andbeta-2 isozyme over the protein kinase C isozymes, alpha, gamma, delta,epsilon, zeta, and eta. In general, the compounds demonstrate a minimumof an eight fold differential in the dosage required to inhibit PKCbeta-1 or beta-2 isozyme and the dosage required for equal inhibition ofthe alpha protein kinase C isozyme as measured in this assay. Therefore,as selective inhibitors of PKC isozyme beta-1 and beta-2, the compoundsare useful in the treatment of conditions in which PKC beta hasdemonstrated a role in the pathology, in particular, diabetes mellitusand its complications.

The following examples are provided merely to further illustrate theinvention. The scope of the invention is not construed as merelyconsisting of the following examples. In the following examples andpreparations, melting point, nuclear magnetic resonance spectra, massspectra, high pressure liquid chromatography over silica gel,N,N-dimethylformamide, palladium on charcoal, diisobutylaluminumhydride, acetonitrile, and tetrahydrofuran are abbreviated M.Pt., NMR,MS, HPLC, DMF, Pd/C, DIBAL, ACN and THF, respectively. The terms "NMR"and "MS" indicate that the spectrum was consistent with the desiredstructure.

EXAMPLE 1 3-[1-(3-azidopropyl)-3-indolyl]-4-[(1-N-^(t) BOC)-glycine-3indolyl]-1H-pyrrol-2,5-dione

3-[1-(3-azidopropyl)-3-indolyl]-4-[3-indolyl]-1H-pyrrol-2,5-dione (0.20g, 0.49 mol, 1 eq) was transfered to an oven dried 50 mL round bottomflask equipped with a stir bar, septum, and N₂ balloon. The red solidwas dissolved in about 25 mL of anhydrous acetonitrile (delivered viacanula) and stirring started. To the red solution was added (N-^(t)BOC)glycine, p-nitrophenyl ester (0.22 g, 0.74 mmol, 1.5 eq), 18-crown-6(0.13 g, 0.49 mmol, 1 eq), and N,N-diisopropylethylamine (0.08 g, 0.61mmol, 1.25 eq, 0.11 mL), and potassium fluoride (0.06 g, 0.98 mmol, 2eq) with vigorous stirring. This red solution was allowed to stir undera N₂ atmosphere for 5 days.

TLC (50% EtOAc in hexanes) of the reaction indicated total loss ofstarting materials. The reaction was diluted with 100 mL of EtOAc,transfered to a separatory funnel, washed with water, and brine. Theorganic layer was dried over MgSO₄. The solvent was removed in vacuo toobtain a red oil. This oil was purified by silica flash chromatographyusing 37.5% EtOAc in hexanes as the mobile phase to give 0.2055 g of anorange solid.

NMR, MS

Elemental Analysis: Theory: C 63.48 N 17.27 H 5.15; Found: C 63.53 N16.28 H 5.64.

EXAMPLE 2 3-[1-(3-N,N'-dimethylaminopropyl)-3-indolyl]-4-[(1-N-^(t)BOC)-glycine-3-indolyl]-1H-pyrrol-2,5-dione

3-[1-(3-N,N'-dimethylaminopropyl)-3-indolyl]-4-[3-indolyl]-1H-pyrrol-2,5-dione(0.05 g, 0.12 mmol, 1 eq) was transfered to a 100 mL round bottom flaskequipped with a stir bar, septum, and N₂ balloon. Anhydrous acetonitrile(70 mL) was delivered via canula followed by pyridine (0.05 g, 0.62mmol, 5 eq, 0.05 mL) via syringe. The resulting orange suspension washeated to 50° C. for 60 minutes to help solubilize the compound. After60 min, (N-^(t) BOC)glycine, p-nitrophenyl ester (0.07 g, 0.24 mmol, 2eq), 18-crown-6 (0.06 g, 0.24 mmol, 2 eq), and dry potassium fluoride(0.03 g, 0.48 mmol, 4 eq) were added as solids with vigorous stirring.The resulting yellow/orange solution was allowed to stir at roomtemperature under an atomosphere of N₂ for 48 hours. MS (FD (in MeOH):MW- 569.67; m/z 570 (MH+)P

EXAMPLE 3 3,4-Bis[(1-N-^(t)BOC)-phenylalanine-3-indolyl]-1H-Pyrrol-2,5-dione

3,4-Bis[3-indolyl]-1H-pyrrol-2,5-dione (0.165 g, 0.5 mmol, 2 eq) wastransfered to an oven dried 50 mL round bottom flask equipped with stirbar, septum, and N₂ balloon. Anhydrous acetonitrile (25 mL) was addedvia canula and stirring started to produce a red solution. (N-^(t)BOC)phenylalanine, p-nitrophenyl ester (0.67 g, 1.75 mmol, 3.5 eq),18-crown-6 (0.13 g, 0.5 mmol, 2 eq), and diisopropylethylamine (0.16 g,1.25 mmol, 2.5 eq, 0.22 mL) were then added to the solution withvigorous stirring. When everything had dissolved, anhydrous potassiumfluoride (0.12 g, 2 mmol, 4 eq) was added as a solid. The solution wasallowed to stir under nitrogen at room temperature overnight.

After 16 hours of stirring, TLC (50% EtOAC in hexanes) showed loss ofstarting material. The yellow solution was transfered to a separatoryfunnel with ˜150 mL of EtOAc. This solution was washed with water andbrine. The organic layer was collected and then dried over MgSO₄. Thesolvent was removed to give a bright yellow/orange residue. This residuewas purified by silica flash chromatography using 37.5% EtOAc in hexanesas the mobile phase to give a bright yellow film after the solvent wasremoved. HPLC of this solid still showed some impurities so the productwas purified using the gel permeation hydrophobic columns with CHC₁₃ asthe mobile phase to give 229 mg of a bright yellow solid. NMR, MS (FD(in MeOH): MW-821.94; m/z 822 (MH+).

EXAMPLE 4 3-[1-(3-acetamidepropyl)-3-indolyl]-4-[(1-N-^(t)BOC)-glycine-3-indolyl]-1H-pyrrol-2,5-dione

3-[1-(3-azidopropyl)-3-indolyl]-4-[(1-N-^(t)BOC)-glycine-3-indolyl]-1H-pyrrol-2,5-dione (80 mg, 0.14 mmol, 1 eq) wasdissolved in ethyl acetate in a 100 mL round bottom flask equipped witha stir bar and 14/22 adapter. Lindlar's catalyst (catalytic, 8 mg) andacetic anhydride (40 mg, 0.39 mmol, 3 eq) were added with vigorousstirring to the red solution. A hydrogen balloon was placed on top ofthe flask. The flask was then evacuated and filled with hydrogen 3 timesto remove dissolved oxygen from the solution. The reaction was allowedto stir at room temperature under the hydrogen atomosphere overnight.

After stirring overnight, TLC (50% EtOAc in hexanes) of the reactionindicated loss of the starting material. The reaction was filteredthrough a pad of celite to remove the hydrogenation catalyst. The redsolution was transfered to a separatory funnel and diluted with ˜100 mLof EtOAc. The organic layer was washed with water and brine and thencollected and dried over MgSO₄. The solvent was removed to give anorange residue. This residue was purified using size exclusion gelpermeation columns using chloroform as the mobile phase to give 52.4 mgof an orange residue. MS (FD (in MeOH): MW-583.65; m/z 584 (MH+),426(M+-amino acid).

    ______________________________________                                                        IC.sub.50 (μm)                                             Ex.  α                                                                              β1                                                                              β2                                                                            γ                                                                             δ                                                                             ε                                                                          ζ                                                                              η                          ______________________________________                                        1    1.4    0.26   0.031                                                                              19    4.6   NA    100  2.6                            2    2.3    0.15   0.04  2.6  2.9   6.1    45  0.37                           3    83     1      2.6  >100  >100  NA   >100  NA                             ______________________________________                                         NA  data not available                                                   

The compounds of Formula I are formulated prior to administration. Apharmaceutical formulation comprises a compound of the Formula I withone or more pharmaceutically acceptable excipients, carriers, ordiluents. Pharmaceutical formulations are prepared by known proceduresusing well known and readily available ingredients. In making thecompositions, the active ingredient will usually be mixed with acarrier, or diluted by a carrier, or enclosed within a carrier, whichmay be in the form of a capsule, sachet, paper or other container. Whenthe carrier serves as a diluent, it may be a solid, semisolid or liquidmaterial which acts as a vehicle, excipient or medium for the activeingredient. Thus, the compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions,solutions, syrups, aerosol (as a solid or in a liquid medium), soft andhard gelatin capsules, suppositories, sterile injectable solutions andsterile packaged powders.

Some examples of suitable carriers, excipients, and diluents includelactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, watersyrup, methyl cellulose, methyl and propylhydroxybenzoates, talc,magnesium stearate and mineral oil. The formulations can additionallyinclude lubricating agents, wetting agents, emulsifying and suspendingagents, preserving agents, sweetening agents or flavoring agents. Thecompositions of the invention may be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient. The compositions are preferablyformulated in a unit dosage form, each dosage containing from about 5 toabout 500 mg, more usually about 25 to about 300 mg, of the activeingredient. However, it will be understood that the therapeutic dosageadministered will be determined by the physician in the light of therelevant circumstances including the condition to be treated, the choiceof compound to be administered and the chosen route of administration,and therefore %he above dosage ranges are not intended to limit thescope of the invention in any way. The term "unit dosage form" refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical carrier.

In addition to the above formulations, the compounds of the presentinvention may be administered topically. Topical formulations areointments, creams, and gels.

Ointments generally are prepared using either (1) an oleaginous base,i.e., one consisting of fixed oils or hydrocarbons, such as whitepetrolatum or mineral oil, or (2) an absorbent base, i.e., oneconsisting of an anhydrous substance or substances that can absorbwater, for example anhydrous lanolin. Customarily, following formationof the base, whether oleaginous or absorbent, the active ingredient(compound) is added to an amount affording the desired concentration.

Creams are oil/water emulsions. They consist of an oil phase (internalphase), comprising typically fixed oils, hydrocarbons, and the like,such as waxes, petrolatum, mineral oil, and the like, and an aqueousphase (continuous phase), comprising water and any water-solublesubstances, such as added salts. The two phases are stabilized by use ofan emulsifying agent, for example, a surface active agent, such assodium lauryl sulfate; hydrophilic colloids, such as acacia colloidalclays, veegum, and the like. Upon formation of the emulsion, the activeingredient (compound) customarily is added to an amount to achieve thedesired concentration.

Gels comprise a base selection from an oleaginous base, water, or anemulsion-suspension base, such as aforedescribed. To the base is added agelling agent that forms matrix in the base increasing its viscosity.Examples of gelling agents are hydroxypropyl cellulose, acrylic acidpolymers, and the like. Customarily, the active ingredient (compounds)is added to the formulation at the desired concentration at a pointpreceding addition of the gelling agent.

The amount of compound incorporated into a topical formulation ofinvention is not critical; the concentration should only be a rangesufficient to permit ready application of the formulation to the anaffected tissue area in an amount that will deliver the desired amountof compound. The customary amount of topical formulation to be appliedto an affected tissue will depend upon an affected tissue size andconcentration of compound in the formulation. Generally, the formulationwill be applied to the an affected tissue in an amount affording fromabout 1 to about 500 μg compound per cm² of an affected tissue.Preferably, the applied amount of compound will range from about 30 toabout 300 μg/cm², more preferably, from about 50 to about 200 μg/cm²,and, most preferably, from about 60 to about 100 μg/cm².

The following formulation examples are illustrative only and are notintended to limit the scope of the invention in any way.

FORMULATION 1

Hard gelatin capsules acre prepared using the following ingredients:

    ______________________________________                                                           Quantity                                                                      (mg/capsule)                                               ______________________________________                                        Active ingredient    250                                                      starch, dried        200                                                      magnesium stearate    10                                                      Total                460 mg                                                   ______________________________________                                    

The above ingredients are mixed and filled into hard gelatin capsules in460 mg quantities.

FORMULATION 2

A tablet is prepared using the ingredients below:

    ______________________________________                                                            Quantity                                                                      (mg/capsule)                                              ______________________________________                                        Active ingredient     250                                                     cellulose, microcrystalline                                                                         400                                                     silicon dioxide, fumed                                                                               10                                                     stearic acid           5                                                      Total                 665 mg                                                  ______________________________________                                    

The components are blended and compressed to form tablets each weighing665 mg.

FORMULATION 3

An aerosol solution is prepared containing the following components:

    ______________________________________                                                        Quantity                                                                      (mg/capsule)                                                  ______________________________________                                        Active ingredient 0.25                                                        ethanol           29.75                                                       Propellant 22     70.00                                                       (chlorodifluoromethane)                                                       Total             100.00                                                      ______________________________________                                    

The active compound is mixed with ethanol. The mixture is added to aportion of the Propellant 22, cooled to -30° C. and transfered to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remainder of the propellant. The valveunits are then fitted to the container.

FORMULATION 4

Tablets each containing 60 mg of active ingredient are made as follows:

    ______________________________________                                                             Quantity                                                                      (mg/capsule)                                             ______________________________________                                        Active ingredient       60 mg                                                 starch                  45 mg                                                 microcrystalline cellulose                                                                            35 mg                                                 polyvinylpyrrolidone                                                          (as 10% solution in water)                                                                            4 mg                                                  sodium carboxymethyl starch                                                                           4.5 mg                                                magnesium stearate      0.5 mg                                                talc                    1 mg                                                  Total                  150 mg                                                 ______________________________________                                    

The active ingredient, starch and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50° C. and passed through a No. 18 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate and talc, previously passedthrough a No. 60 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 150 mg.

FORMULATION 5

Capsules each containing 80 mg of medicament are made as follows:

    ______________________________________                                                         Quantity                                                                      (mg/capsule)                                                 ______________________________________                                        Active ingredient  80 mg                                                      starch             59 mg                                                      microcrystalline cellulose                                                                       59 mg                                                      magnesium stearate  2 mg                                                      Total              200 mg                                                     ______________________________________                                    

The active ingredient, cellulose, starch and magnesium stearate areblended, passed through a No. 45 mesh U.S. sieve, and filled into hardgelatin capsules in 200 mg quantities.

FORMULATION 6

Suppositories each containing 225 mg of active ingredient may be made asfollows:

    ______________________________________                                                          Quantity                                                                      (mg/capsule)                                                ______________________________________                                        Active ingredient     225 mg                                                  saturated fatty acid glycerides                                                                   2,000 mg                                                  Total               2,225 mg                                                  ______________________________________                                    

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

FORMULATION 7

Suspensions each containing 50 mg of medicament per 5 ml dose are madeas follows:

    ______________________________________                                                             Quantity                                                                      (mg/capsule)                                             ______________________________________                                        Active ingredient      50 mg                                                  sodium carboxymethyl cellulose                                                                       50 mg                                                  syrup                   1.25 ml                                               benzoic acid solution   0.10 ml                                               flavor                 q.v.                                                   color                  q.v.                                                   purified water to total                                                                               5 ml                                                  ______________________________________                                    

The medicament is passed through a No. 45 mesh U.S. sieve and mixed withthe sodium carboxymethyl cellulose and syrup to form a smooth paste. Thebenzoic acid solution, flavor and color are diluted with some of thewater and added, with stirring. Sufficient water is then added toproduce the required volume.

FORMULATION 8

An intravenous formulation may be prepared as follows:

    ______________________________________                                                      Quantity                                                                      (mg/capsule)                                                    ______________________________________                                        Active ingredient                                                                              250 mg                                                       isotonic saline 1000 mg                                                       ______________________________________                                    

The solution of the above ingredients is administered intravenously at arate of 1 ml per minute to a subject in need of treatment.

We claim:
 1. A compound of the formula: ##STR12## wherein: R₁ is of theformula: ##STR13## R₂ is hydrogen, alkyl, R₃ is H or CH₃ ;R₄ is an aminoacid side chain; X is --(CH₂)_(n) --NH--, --(CH₂)_(n) --O--,phenylene--NH--, phenylene--O--, or a bond; P₁ is H, alkyl, or an aminoprotecting group; and n is 1, 2 or 3; or a pharmaceutically acceptablesalt or solvate thereof.
 2. A compound off claim 1 wherein X is a bond.3. A compound off claim 2 wherein R₃ is H.
 4. A compound of claim 3 ofthe formula: ##STR14## wherein R₂ is hydrogen or alkyl;R₄ is H, CH₃,##STR15## and P₁ is H, t-butoxycarbonyl, or benzyloxycarbonyl.
 5. Amethod for treating diabetes mellitus, which comprises administering toa mammal in need of such treatment a pharmaceutically effective amountof a compound of claim
 1. 6. A method for treating diabetes mellitus,which comprises administering to a mammal in need of such treatment apharmaceutically effective amount of a compound of claim
 2. 7. A methodfor treating diabetes mellitus, which comprises administering to amammalin need of such treatment a pharmaceutically effective amount of acompound of claim
 3. 8. A method for treating diabetes mellitus, whichcomprises administering to a mammal in need of such treatment apharmaceutically effective amount of a compound of claim
 4. 9. A methodof selectively inhibiting protein kinase C beta-1 and beta-2 isozyme,which comprises administering to a mammal in need of such treatment apharmaceutically effective amount of a compound of claim
 1. 10. A methodof selectively inhibiting protein kinase C beta-1 and beta-2 isozyme,which comprises administering to a mammal in need of such treatment apharmaceutically effective amount of a compound of claim
 2. 11. A methodof selectively inhibiting protein kinase C beta-1 and beta-2 isozyme,which comprises administering to a mammal in need of such treatment apharmaceutically effective amount of a compound of claim
 3. 12. A methodof selectively inhibiting protein kinase C beta-1 and beta-2 isozyme,which comprises administering to a mammal in need of such treatment apharmaceutically effective amount of a compound of claim
 4. 13. Apharmaceutical formulation comprising a compound of claim 1 with one ormore pharmaceutically acceptable excipients, carriers, or diluents. 14.A pharmaceutical formulation comprising a compound of claim 2 with oneor more pharmaceutically acceptable excipients, carriers, or diluents.15. A pharmaceutical formulation comprising a compound of claim 3 withone or more pharmaceutically acceptable excipients, carriers, ordiluents.
 16. A pharmaceutical formulation comprising a compound ofclaim 4 with one or more pharmaceutically acceptable excipients,carriers, or diluents.